Halogenated organic compounds are used extensively as building blocks, synthetic intermediates, and end use products for pharmaceutical and agrochemical applications. The utility of these compounds, including their biological activity, arises from the reactivity and physical properties uniquely conferred to them by halogen substitution. The importance of halogenation and limitations associated with current halogenation methods prompted us to develop new halogenation catalysts. This proposal outlines the evolution of flavin dependent halogenases (FDHs) for a range synthetic methods involving selective halogenation. Our preliminary results include the first examples of FDH directed evolution via iterative mutagenesis, and we will use the methods we have developed to further improve the substrate scope, selectivity, and reactivity of these enzymes for practical synthetic applications. This will entail generating FDH variants and screening those variants for activity on substrates with progressively increased sizes, altered shapes, and reduced nucleophilicities, relative to the native substrate, tryptophan. Structural, kinetic, and computational methods will then be used to provide rigorous characterization of the engineered enzymes. These data will inform subsequent engineering efforts. Specifically, FDHs will be engineered to override substrate reactivity using molecular recognition and to tune the biological activity of arene and olefin-containing substrates via late-stage halogenation and tandem halogenation/cross-coupling. Synthesis of PET probes using FDH catalyzed incorporation of positron emitting halogens into these compounds will also be demonstrated. Together, these experiments will significantly advance modern synthetic halogenation methods and improve our ability to engineer enzymes for other C-H bond functionalization reactions.